Synthesis of human bone morphogenetic protein-2 (hBMP-2) in E. coli periplasmic space: its characterization and preclinical testing
Cells, v. 10, n. 12, 3525, dez. 2021
Article
en Pt
| SES-SP, SESSP-IBPROD, SES-SP
| ID: bud-4086
Biblioteca responsable:
BR78.1
RESUMO
Human BMP-2, a homodimeric protein that belongs to the TGF- β family, is a recognized osteoinductor due to its capacity of inducing bone regeneration and ectopic bone formation. The administration of its recombinant form is an alternative to autologous bone grafting. A variety of E. coli-derived hBMP-2 has been synthesized through refolding of cytoplasmic inclusion bodies. The present work reports the synthesis, purification, and characterization of periplasmic hBMP-2, obtained directly in its correctly folded and authentic form, i.e., without the initial methionine typical of the cytoplasmic product that can induce undesired immunoreactivity. A bacterial expression vector was constructed including the DsbA signal peptide and the cDNA of hBMP-2. The periplasmic fluid was extracted by osmotic shock and analyzed via SDS-PAGE, Western blotting, and reversed-phase high-performance liquid chromatography (RP-HPLC). The purification was carried out by heparin affinity chromatography, followed by high-performance size-exclusion chromatography (HPSEC). HPSEC was used for qualitative and quantitative analysis of the final product, which showed >95% purity. The classical in vitro bioassay based on the induction of alkaline phosphatase activity in myoblastic murine C2C12 cells and the in vivo bioassay consisting of treating calvarial critical-size defects in rats confirmed its bioactivity, which matched the analogous literature data for hBMP-2.
Texto completo:
1
Colección:
06-national
/
BR
Base de datos:
SES-SP
/
SESSP-IBPROD
Tipo de estudio:
Qualitative_research
Idioma:
Pt
Revista:
Cells
Año:
2021
Tipo del documento:
Article